For purposes of this specification, the term "gene" or "genes" is used to mean nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein. Genes that are not part of a particular plant's genome are referred to as "foreign genes" and genes that are a part of a particular plant's genome are referred to as "endogenous genes". The term "gene products" refers to RNAs or proteins that are encoded by the gene. "Foreign gene products" are RNA or proteins encoded by foreign genes and "endogenous gene products" are RNA or proteins encoded by endogenous genes.
It has been known for some time that plants may be used to express foreign gene products, or to overexpress endogenous gene products, via introduction of the foreign or endogenous gene into a plant through the use of various biotechnological methods. In one biotechnological approach, the gene encoding the gene product of interest is introduced into the plant genome under control of a promoter sequence that is functional in the plant, resulting in the transcription of the gene to produce messenger RNA, followed by various RNA processing events, exit of the RNA from the nucleus and translation of the messenger RNA to produce the encoded protein. This approach has been exploited by agricultural and industrial interests to provide a ready and relatively inexpensive source of a variety of beneficial gene products.
In some cases the gene products serve their function in the plant from which they are expressed. A natural insecticide that confers resistance to insects in commercially available transgenic crop plants is one such example. In other cases, the gene product of interest may be extracted from the plant and serves its function elsewhere. For example, potentially valuable proteins, such as antibodies, may be expressed in plants. Such production methods are seen as a marked improvement over the use of animal tissue for such production.
Several years after the first series of publications detailing methods to introduce foreign genes or additional copies of endogenous genes into plants, an unexplained phenomenon was reported. Plants containing an additional copy of an endogenous plant gene not only failed to display the hoped for enhancement in the accumulation of the gene product, but also repressed expression of the endogenous gene, effectively eliminating the expression of the endogenous gene product. This phenomenon was referred to as "cosuppression" since expression of both the endogenous gene and the introduced transgene were suppressed. The incidence of cosuppression in transformed plants containing extra copies of an endogenous gene is high. Up to 90% of independently transformed petunia plants containing an introduced chalcone synthase gene show some variegation in petal color, which is indicative of cosuppression of this gene. It has been postulated that this same type of gene expression suppression may occur whenever a particular messenger RNA sequence is expressed at high levels. This may explain the generally low levels of expression of introduced genes in plants.
Whatever the explanation, the inactivation of gene expression by cosuppression is a problem in cases where high levels of expression of an introduced gene or over expression of an endogenous gene is desirable. Thus, the use of transgenic plants to express introduced genes has been limited due to this general constraint on high level gene expression in the plant cells.
Another approach to expressing foreign or endogenous gene products in plants is the use of plant viruses as vectors to express foreign genes in an appropriate host plant. One example of a viral vector for expressing a foreign gene is described in U.S. Pat. Nos. 5,316,931 and 5,589,367, both naming Donson et al. as inventors. Both of these patents are incorporated in their entireties herein by reference. These patents provide recombinant plant viral nucleic acids and recombinant viruses that are stable for maintenance and transcription or expression of non-native (foreign) nucleic acid sequences and which are capable of systemically transcribing or expressing the foreign sequences in the host plant.
Others have also attempted to use various viral-based vectors to express genes that are not native to the virus. For example, Takamatsu et al. described the use of tobacco mosaic virus ("TMV") as a vector to express enkephalin in "Production of Enkephalin in Tobacco Protoplasts Using Tobacco Mosaic Virus RNA Vector," 269 FEBS Lett., 73-76 (1990). In 1993, Hamamoto et al. described the production of an angiotensin-I-converting enzyme inhibitor peptide from a TMV RNA vector in "A New Tobacco Mosaic Virus Vector and Its Use for the Systemic Production of Angiotensin-I-Converting Inhibitor in Transgenic Tobacco and Tomato," 11 Bio/Technology, 930-932 (1993). Kumagai, et al. disclosed using a tobamovirus as a viral vector to produce an HIV-inhibitor, .alpha.-Trichosanthin in "Rapid, High-Level Expression of Biologically Active .alpha.-Trichosanthin in Transfected Plants by an RNA-Viral Vector," 90 Proc. Natl. Acad. Sci. USA, 427-430 (1993).
Other examples of using viral vectors to express foreign gene products by various methods are known to those of skill in the art. Generally, suitable plant viral vectors for expressing foreign genes should be self-replicating, capable of systemic infection in a host, and stable. In addition, they should be capable of containing the nucleic acid sequences that are foreign to the native virus forming the vector.
Although using plant viruses to express foreign gene products generally allows expression of the products at a higher level than that obtained from genes introduced stably in the plant genome, current methods of expressing genes from viral vectors suffer from several practical limitations. The virus is often debilitated when a foreign gene is cloned into it. When a foreign gene sequence (one not native to the virus vector) is introduced into a virus, the virus is weakened and the weakened virus does not produce its gene products as readily. In addition to debilitation of viral gene expression, the virus is unable to replicate and move as efficiently through the host plant as can wild-type parental viruses. Furthermore, viruses carrying foreign genes tend to be unstable and often delete the inserted genes as the viruses replicate. These tendencies are discussed in Dolia, et al., "Tagging of Plant Potyvirus Replication and Movement by Insertion of .beta.-Glucuronidase into the Viral Polyprotein," 89 Proc. Natl. Acad. Sci. USA, 10208-10212 (1992) and Chapman et al., "Potato Virus X as a Vector for Gene Expression in Plants, 2 The Plant Journal, 549-557 (1992).
It has also been known for some time that in plants infected with more than one virus at the same time, the two co-infecting viruses may interact synergistically to cause a more severe disease in the plant than does either virus alone. In many cases it has been shown that the increase in severity of host symptoms correlates with an increase in the accumulation of one virus of the synergistic pair. For example, it is known that a synergistic disease is caused by the interaction of potato virus X ("PVX") and potato virus Y ("PVY"). PVX in such synergistically-diseased plants accumulates to a higher level than in singly infected plants and eventually causes the first systemically infected leaves of the doubly infected plant to die. The infection by either PVX or PVY alone in the same plant has little or no effect at all.
These synergistic effects have also been demonstrated as a result of PVX interaction with at least three other members of the potyvirus group of plant viruses,-tobacco vein mottling virus ("TVMV"), tobacco etch virus ("TEV"), and pepper mottle virus ("PepMoV"). Such PVX/potyvirus mixed infections of a tobacco host plant result in a dramatic increase in accumulation of PVX particles (up to ten-fold), with no corresponding increase or decrease in accumulation of the potyvirus particles. These PVX/potyviral mixed infections also result in a dramatic increase in disease symptoms in the doubly infected plant.
The initial step in the present discovery of the viral booster sequence was the finding that the PVX/potyviral synergistic disease syndrome, characterized by increases in symptom severity and in accumulation of the PVX pathogen, does not require infection with both viruses. This was reported by Vance, et al. in "5' Proximal Potyviral Sequences Mediate Potato Virus X/Potyviral Synergistic Disease in Transgenic Tobacco," 206 Virology, 583-590 (1995). The synergistic disease is mimicked in plants expressing only a subset of the potyviral genomic RNA and infected singly with PVX. The potyviral region shown to mediate the synergistic disease comprises the 5'-proximal 2780 nucleotides of the genomic RNA, including the 5'-untranslated region (5'-UTR) and the region encoding the potyviral gene products P1, helper component-proteinase (HC-Pro) and a portion of P3. This described potyviral region is referred to herein as the "P1/HC-Pro sequence".
Thus, Vance, et al. (1995), identified a disease determinant carried by the potyvirus genome (the P1/HC-Pro sequence), and this disease determinant was shown to mediate the well-known PVX/potyviral synergistic disease. Although the mechanism by which this potyviral sequence mediated the PVX/potyviral synergistic disease was unknown, it was postulated to involve a specific, direct interaction of the potyviral P1/HC-Pro RNA sequence or the encoded potyviral gene products with the genomic RNA or replication proteins of the interacting PVX pathogen. Although the potyviral P1/HC-Pro sequence was found to boost accumulation of the PVX viral structural gene (coat protein) and the accumulation of the PVX viral particle, this enhanced accumulation was thought to be specific for the native PVX genes expressed from the native PVX genome. Furthermore, the enhanced accumulation of PVX coat protein and PVX virus particles was tightly correlated with the perceived detrimental and undesirable increase in disease symptoms.
Although PVX and other viruses, such as cauliflower mosaic virus, the geminiviruses, and TMV, have been used as viral vectors for expressing foreign gene products, such vectors have not been completely successful as mentioned above. Although gene products have been produced via expression from viral vectors, the usefulness of such vectors is limited by instability of inserted sequences and the failure of the viral vector to replicate efficiently. Expression of gene products from genes introduced stably into the plant genome also suffers from limitations as mentioned above.
Accordingly, it would be beneficial if methods of producing gene products from plants could be developed to allow enhanced expression of the introduced gene from stably transformed plants and enhanced expression of genes introduced into a plant via a plant viral vector, while retaining stability of the introduced sequence to ensure accumulation of the gene product. The present invention overcomes some of the deficiencies of prior gene product expression methods by using a particular boosting sequence obtained from a potyvirus.